The diagnosis, prognosis, and determination of treatment of disease based on the interpretation of tissue or cell samples taken from a diseased organism has expanded dramatically over the past few years. In addition to traditional histological staining techniques and immunohistochemical assays, in situ techniques such as in situ hybridization and in situ polymerase chain reaction are now used to help diagnose disease states in humans. Thus, there are a variety of techniques that can assess not only cell morphology, but also the presence of specific macromolecules within cells and tissues.
Molecular cytogenetic techniques, such as chromogenic in situ hybridization (CISH) combine visual evaluation of chromosomes (karyotypic analysis) with molecular techniques. Molecular cytogenetics methods are based on hybridization of a nucleic acid probe to its complementary nucleic acid within a cell. A probe for a specific chromosomal region will recognize and hybridize to its complementary sequence on a metaphase chromosome or within an interphase nucleus (for example in a tissue sample). Probes have been developed for a variety of diagnostic and research purposes.
Sequence probes hybridize to single copy DNA sequences in a specific chromosomal region or gene. These are the probes used to identify the chromosomal critical region or gene associated with a syndrome or condition of interest. On metaphase chromosomes, such probes hybridize to each chromatid, usually giving two small, discrete signals per chromosome.
Hybridization of sequence probes, such as repeat depleted probes or unique sequence probes (see for example U.S. 2011/0160076, which is hereby incorporated by reference in its entirety for disclosure related to unique sequence probes), has made possible detection of chromosomal abnormalities associated with numerous diseases and syndromes, including constitutive genetic anomalies, such as microdeletion syndromes, chromosome translocations, gene amplification and aneuploidy syndromes, neoplastic diseases as well as pathogen infections. Most commonly these techniques are applied to standard cytogenetic preparations on microscope slides. In addition, these procedures can be used on slides of formalin-fixed paraffin embedded tissue, blood or bone marrow smears, and directly fixed cells or other nuclear isolates.
The information obtained from these assays can be used to diagnose disease in a patient, determine the prognosis of a patient that has a disease, and also to determine the course of treatment for a patient with a disease. In many instances, the presence of a particular marker can be associated with the predicted efficacy of a drug.
Non-small cell lung cancer (NSCLC) is a disease in which malignant (cancer) cells form in the tissues of the lung. NSCLC is actually a group of lung cancers that are named for the kinds of cells found in the cancer and how the cells look under a microscope. The three main types of non-small cell lung cancer are squamous cell carcinoma, large cell carcinoma, and adenocarcinoma. NSCLC is the most common kind of lung cancer.
Squamous cell carcinoma is a cancer that begins in squamous cells, which are thin, flat cells that look like fish scales. This is also called epidermoid carcinoma. Large cell carcinoma is a cancer that may begin in several types of large cells. Adenocarcinoma is a cancer that begins in the cells that line the alveoli and make substances such as mucus. Other less common types of non-small cell lung cancer are: pleomorphic, carcinoid tumor, salivary gland carcinoma, and unclassified carcinoma.
Smoking cigarettes, pipes, or cigars is the most common cause of NSCLC. The earlier in life a person starts smoking, the more often a person smokes, and the more years a person smokes, the greater the risk. If a person has stopped smoking, the risk becomes lower as the years pass.
Tests and procedures to detect, diagnose, and stage non-small cell lung cancer are often done at the same time. The following tests and procedures are generally used: Chest x-ray; CBC; Sputum test to look for cancer cells; Bone scan; CT scan of the chest; MRI of the chest; Positron emission tomography (PET) scan; and Thoracentesis. In some instances, biopsies are taken and analyzed. If the biopsy reveals the presence of lung cancer, more imaging tests will be done to determine the stage of the cancer. Stage relates to the size of the tumor and the extent to which it has spread. Non-small cell lung cancer is divided into five stages: Stage 0— the cancer has not spread beyond the inner lining of the lung; Stage I—the cancer is small and has yet to spread to the lymph nodes; Stage II—the cancer has spread to some lymph nodes near the original tumor; Stage III—the cancer has spread to nearby tissue or spread to far away lymph nodes; Stage IV—the cancer has spread to other organs of the body such as the other lung, brain, or liver.
There are many different types of treatment for non-small cell lung cancer. Treatment depends upon the stage of the cancer. Surgery is the often the first line of treatment for patients with non-small cell lung cancer that has not spread beyond nearby lymph nodes. The surgeon may remove: One of the lobes of the lung (lobectomy); only a small part of the lung (wedge or segment removal); the entire lung (pneumonectomy). Some patients need chemotherapy. Chemotherapy uses drugs to kill cancer cells and stops new ones from growing. Chemotherapy alone is often used when the cancer has spread (stage IV).
In some instances, a genetic analysis is done to determine the best course of treatment for NSCLC. For example, some patients with particular mutations in the EGFR gene respond to EGFR tyrosine kinase inhibitors such as gefitinib. As another example, the 7% of NSCLC with EML4-ALK translocations may benefit from ALK inhibitors which are in clinical trials.
Break-apart probe systems have been used for analysis of tissues from NSCLC patients. However, due to the nature of the chromosomal rearrangements that occur in NSCLC, there can be a problem with false positive results, especially where the rearrangement is within the same chromosome, such as an inversion. In these cases, it may not be possible to properly resolve the signals from each set of break-apart probes. The signals can appear as two separate signals even though no rearrangement has occurred. This can be a real problem, both due to obtaining incorrect results and the scarcity of biopsy material. Three color systems have been used for chromosomal analysis. See, e.g., Makretsov et al., Genes, Chromosomes and Cancer, 40:152-57 (2004); Martin-Subero, et al., Cancer Res., 66(21):10332-38 (2006); Yoshimoto et al., Neoplasia 8(6):465-69 (2006); Renne et al., J. Mol. Diagnost., 7(3): 352-56 (2005). However, none of these systems have been applied to solve problems associated with false positive results in break-apart probe systems. Break-apart probe systems which address the problem of false positive results would provide a benefit to patients afflicted with cancer.